Enhanced levels of vitamin B(6) increase aerial organ size and positively affect stress tolerance in Arabidopsis

Vitamin B₆ is an essential nutrient in the human diet derived primarily from plant sources. While it is well established as a cofactor for numerous metabolic enzymes, more recently, vitamin B₆ has been implicated as a potent antioxidant. The de novo vitamin B₆ biosynthesis pathway in plants has recently been unraveled and involves only two proteins, PDX1 and PDX2. To provide more insight into the effect of the compound on plant development and its role as an antioxidant, we have overexpressed the PDX proteins in Arabidopsis, generating lines with considerably higher levels of the vitamin in comparison with other recent attempts to achieve this goal. Interestingly, it was possible to increase the level of only one of the two catalytically active PDX1 proteins at the protein level, providing insight into the mechanism of vitamin B₆ homeostasis in planta. Vitamin B₆ enhanced lines have considerably larger vegetative and floral organs and although delayed in pre‐reproductive development, do not have an altered overall morphology. The vitamin was observed to accumulate in seeds and the enhancement of its levels was correlated with an increase in their size and weight. This phenotype is predominantly a consequence of embryo enlargement as reflected by larger cells. Furthermore, plants that overaccumulate the vitamin have an increased tolerance to oxidative stress providing in vivo evidence for the antioxidant functionality of vitamin B₆. In particular, the plants show an increased resistance to paraquat and photoinhibition, and they attenuate the cell death response observed in the conditional flu mutant.     

Effects of vitamin B6 supplementation in rats during lactation on vitamin B6 concentration and transaminase activities in the offspring

The aim of the present investigation was to study the effect of a varying maternal vitamin B₆ supplementation during lactation period on vitamin B₆ levels in blood, liver and total body, and on the activity of two transaminase enzymes in the offspring. Therefore, eighty female Sprague‐Dawley rats were fed a semi‐synthetic diet (0.2 mg vitamin B₆ per kg) which was supplemented during gravidity with 5 mg vitamin B₆ per kg diet. During the following lactation period the rats were assigned to one of 10 vitamin B₆ treatment groups (supplementation of 0, 3, 6, 9, 12, 15, 18, 36, 360, 3600 mg vitamin B₆ per kg diet). At day 14 of lactation the pubs of all dams were decapitated and blood, liver, and carcass were used for analysis of vitamin B₆ concentration, activities of two transaminases, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in plasma, erythrocytes, and liver, and of haematological parameters.

While the liver and total body wet weights as well as the haematological parameters (red blood cells, haemoglobin concentration, hematocrit, middle corpuscular cell volume, middle corpuscular haemoglobin, middle corpuscular haemoglobin concentration) did not differ within the experimental groups, the present data clearly show that in blood, liver and total body of the offspring exists a slight dose‐response relationship between the maternal dietary vitamin B₆ supplementation and the vitamin B₆ concentration. Concerning the activities of the transaminases a dietary supplementation above 3mg vitamin B₆ per kg diet had no influence on the AST and ALT activities in offspring plasma. In the erythrocytes no statistical significant influence of the vitamin B₆ supplementation during lactation on the activities of AST and ALT was found. The activities of ALT and AST in liver were not consistently altered by the vitamin B₆ supplementation of the dams during lactation. In conclusion these results indicate that a minimal maternal dietary vitamin B₆ supply of 3.1 mg per kg diet is necessary with regard to health and development of their offspring. But not all of the analysed parameters as the liver and total body weights, the activities of AST and ALT in the erythrocytes, and the haematological parameters were influenced by a deficient maternal dietary vitamin B₆ supply.     

The Pseudoenzyme PDX1.2 Boosts Vitamin B6 Biosynthesis under Heat and Oxidative Stress in Arabidopsis

Vitamin B₆ is an indispensable compound for survival, well known as a cofactor for numerous central metabolic enzymes and more recently for playing a role in several stress responses, particularly in association with oxidative stress. Regulatory aspects for the use of the vitamin in these roles are not known. Here we show that certain plants carry a pseudoenzyme (PDX1.2), which is involved in regulating vitamin B₆ biosynthesis de novo under stress conditions. Specifically, we demonstrate that Arabidopsis PDX1.2 enhances the activity of its catalytic paralogs by forming a heterododecameric complex. PDX1.2 is strongly induced by heat as well as singlet oxygen stress, concomitant with an enhancement of vitamin B₆ production. Analysis of pdx1.2 knockdown lines demonstrates that boosting vitamin B₆ content is dependent on PDX1.2, revealing that this pseudoenzyme acts as a positive regulator of vitamin B₆ biosynthesis during such stress conditions in plants.     

Rice plastidial NAD‐dependent malate dehydrogenase 1 negatively regulates salt stress response by reducing the vitamin B6 content

Salinity is an important environmental factor that adversely impacts crop growth and productivity. Malate dehydrogenases (MDHs) catalyse the reversible interconversion of malate and oxaloacetate using NAD(H)/NADP(H) as a cofactor and regulate plant development and abiotic stress tolerance. Vitamin B6 functions as an essential cofactor in enzymatic reactions involved in numerous cellular processes. However, the role of plastidial MDH in rice (Oryza sativa) in salt stress response by altering vitamin B6 content remains unknown. In this study, we identified a new loss‐of‐function osmdh1 mutant displaying salt stress‐tolerant phenotype. The OsMDH1 was expressed in different tissues of rice plants including leaf, leaf sheath, panicle, glume, bud, root and stem and was induced in the presence of NaCl. Transient expression of OsMDH1‐GFP in rice protoplasts showed that OsMDH1 localizes to chloroplast. Transgenic rice plants overexpressing OsMDH1 (OsMDH1OX) displayed a salt stress‐sensitive phenotype. Liquid chromatography–mass spectrometry (LC‐MS) metabolic profiling revealed that the amount of pyridoxine was significantly reduced in OsMDH1OX lines compared with the NIP plants. Moreover, the pyridoxine content was higher in the osmdh1 mutant and lower in OsMDH1OX plants than in the NIP plants under the salt stress, indicating that OsMDH1 negatively regulates salt stress‐induced pyridoxine accumulation. Furthermore, genome‐wide RNA‐sequencing (RNA‐seq) analysis indicated that ectopic expression of OsMDH1 altered the expression level of genes encoding key enzymes of the vitamin B6 biosynthesis pathway, possibly reducing the level of pyridoxine. Together, our results establish a novel, negative regulatory role of OsMDH1 in salt stress tolerance by affecting vitamin B6 content of rice tissues.     

Balancing of B6 Vitamers Is Essential for Plant Development and Metabolism in Arabidopsis

Vitamin B₆ comprises a family of compounds that is essential for all organisms, most notable among which is the cofactor pyridoxal 5′-phosphate (PLP). Other forms of vitamin B₆ include pyridoxamine 5′-phosphate (PMP), pyridoxine 5′-phosphate (PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also have salvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways to cellular vitamin B₆ homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensively characterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNP oxidase PDX3 is essential for balancing B₆ vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth and development are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction of stress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acid levels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction in nitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an important link between vitamin B₆ homeostasis and nitrogen metabolism.     

Life's utilization of B vitamins on early Earth

Coenzymes are essential across all domains of life. B vitamins (B₁‐thiamin, B₂‐riboflavin, B₃‐niacin, B₅‐pantothenate, B₆‐pyridoxine, B₇‐biotin, and B₁₂‐cobalamin) represent the largest class of coenzymes, which participate in a diverse set of reactions including C₁‐rearrangements, DNA repair, electron transfer, and fatty acid synthesis. B vitamin structures range from simple to complex heterocycles, yet, despite this complexity, multiple lines of evidence exist for their ancient origins including abiotic synthesis under putative early Earth conditions and/or meteorite transport. Thus, some of these critical coenzymes likely preceded life on Earth. Some modern organisms can synthesize their own B vitamins de novo while others must either scavenge them from the environment or establish a symbiotic relationship with a B vitamin producer. B vitamin requirements are widespread in some of the most ancient metabolisms including all six carbon fixation pathways, sulfate reduction, sulfur disproportionation, methanogenesis, acetogenesis, and photosynthesis. Understanding modern metabolic B vitamin requirements is critical for understanding the evolutionary conditions of ancient metabolisms as well as the biogeochemical cycling of critical elements such as S, C, and O.     

Mutualistic interactions between vitamin B(12) -dependent algae and heterotrophic bacteria exhibit regulation

Many algae are auxotrophs for vitamin B₁₂ (cobalamin), which they need as a cofactor for B₁₂‐dependent methionine synthase (METH). Because only prokaryotes can synthesize the cobalamin, they must be the ultimate source of the vitamin. In the laboratory, a direct interaction between algae and heterotrophic bacteria has been shown, with bacteria supplying cobalamin in exchange for fixed carbon. Here we establish a system to study this interaction at the molecular level. In a culture of a B₁₂‐dependent green alga Chlamydomonas nivalis, we found a contaminating bacterium, identified by 16S rRNA analysis as Mesorhizobium sp. Using the sequenced strain of M. loti (MAFF303099), we found that it was able to support the growth of B₁₂‐dependent Lobomonas rostrata, another green alga, in return for fixed carbon. The two organisms form a stable equilibrium in terms of population numbers, which is maintained over many generations in semi‐continuous culture, indicating a degree of regulation. However, addition of either vitamin B₁₂ or a carbon source for the bacteria perturbs the equilibrium, demonstrating that the symbiosis is mutualistic and facultative. Chlamydomonas reinhardtii does not require B₁₂ for growth because it encodes a B₁₂‐independent methionine synthase, METE, the gene for which is suppressed by addition of exogenous B₁₂. Co‐culturing C. reinhardtii with M. loti also results in reduction of METE expression, demonstrating that the bacterium can deliver the vitamin to this B₁₂‐independent alga. We discuss the implications of this for the widespread distribution of cobalamin auxotrophy in the algal kingdom.     

Identification of a second pyridoxine (pyridoxamine) 5′-phosphate oxidase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Pyridoxine (pyridoxamine) 5′-phosphate oxidase (PPOX) is involved in the biosynthetic pathway of vitamin B₆, converting pyridoxine 5′-phosphate (PNP) or pyridoxamine 5′-phosphate (PMP) into pyridoxal 5′-phosphate (PLP). PLP is a well-known cofactor of numerous enzymes including transamination and decarboxylation reactions. We have previously identified a PPOX (AtPPOX-1) protein encoded by At5g49970 in Arabidopsis thaliana. Here, we report a second PPOX in Arabidopsis, which was named as AtPPOX-2 encoded by At2g46580. The RT-PCR amplified cDNA of AtPPOX-2 was cloned into an Escherichia coli expression vector and a yeast shuttle vector. Both PPOX enzyme assay and complementation of the oxidative stress sensitivity phenotype of a yeast PDX3 deletion mutant demonstrated that At2g46580 encodes a PPOX protein (AtPPOX-2). The catalytic efficiency of AtPPOX-1 is approximately 300-fold higher than that of AtPPOX-2 for PNP. Based on bioinformatic analysis, AtPPOX-2 has a putative mitochondrial transit peptide at the N-terminus. The truncated AtPPOX-2 without 18 amino acids at the N-terminal end lost PPOX activity, suggesting that the N-terminal 18 amino acids are necessary for the enzyme activity of AtPPOX-2. Phylogenetic analysis of AtPPOX-2 homologs from all domains of life suggests that AtPPOX-2 homologs in plants are the product of lateral gene transfer from the cyanobacterial endosymbionts from which plastids are derived.     

Expression of γ‐tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal‐induced oxidative stresses by reducing reactive oxygen species

The γ‐tocopherol methyltransferase (γ‐TMT) is an important enzyme regulating synthesis of four tocopherols (α, γ, β and δ). In this report, we investigated the role of γ‐TMT in regulating abiotic stress within chloroplasts. The At γ‐tmt overexpressed via the tobacco chloroplast genome accumulated up to 7.7% of the total leaf protein, resulting in massive proliferation of the inner envelope membrane (IEM, up to eight layers). Such high‐level expression of γ‐TMT converted most of γ‐tocopherol to α‐tocopherol in transplastomic seeds (~10‐fold higher) in the absence of abiotic stress. When grown in 400 mm NaCl, α‐tocopherol content in transplastomic TMT leaves increased up to 8.2‐fold and 2.4‐fold higher than wild‐type leaves. Likewise, under heavy metal stress, α‐tocopherol content in the TMT leaves increased up to 7.5‐fold, twice higher than in the wild type. Under extreme salt stress, the wild type accumulated higher starch and total soluble sugars, but TMT plants were able to regulate sugar transport. Hydrogen peroxide and superoxide content in wild type increased up to 3‐fold within 48 h of NaCl stress when compared to TMT plants. The ion leakage from TMT leaves was significantly less than wild‐type plants under abiotic stress and with less malondialdehyde, indicating lower lipid peroxidation. Taken together, these studies show that α‐tocopherol plays a crucial role in the alleviation of salt and heavy metal stresses by decreasing ROS, lipid peroxidation and ion leakage, in addition to enhancing vitamin E conversion. Increased proliferation of the IEM should facilitate studies on retrograde signalling from chloroplast to the nucleus.     

Structure, evolution and action of vitamin B6-dependent enzymes

The number of known three-dimensional structures of vitamin B₆-dependent enzymes has doubled in the past two years. A fourth type of fold for B₆-dependent enzymes, involving a TIM-barrel domain, has been discovered. Alanine racemase is the first known representative of this new fold. Significant progress has been made in understanding the allosteric effects in the tryptophan synthase reaction.     

Enhancement of vitamin B6 levels in seeds through metabolic engineering

As a versatile cofactor for many enzymes catalyzing important biochemical reactions, vitamin B₆ is required for all cellular organisms. In contrast to bacteria, fungi and plants, which have the ability to synthesize vitamin B₆ de novo , animals have to take up the vitamin from their diet. Plants are the major source of vitamin B₆ for animals. The recent identification of vitamin B₆ biosynthetic enzymes PDX1 and PDX2 in plants makes it possible to regulate the biosynthesis of this important vitamin. In this study, we generated Arabidopsis plants overexpressing the PDX1 and/or PDX2 gene and used a liquid chromatography/mass spectrometry/mass spectrometry method to determine the levels of different forms of vitamin B₆ in these transgenic plants. It was found that expression of the PDX genes under control of the CaMV 35S promoter caused only a limited increase in pyridoxine contents in dry seeds but not in shoots or roots. When using the Arabidopsis seed-specific 12S promoter to drive the expression of the PDX genes, the levels of vitamin B₆ increased more than twofold in transgenic plants. Our work demonstrates that it is feasible to enhance vitamin B₆ content in seeds by metabolic engineering.     

The nature of the requirement of Saccharomyces carlsbergensis for vitamin B6

Saccharomyces carlsbergensis 4228, an organism widely used for determination of vitamin B₆, grows well without this vitamin if thiamine is also omitted from the basal medium, and an inoculum grown in a thiamine-low medium is used. Thiamine inhibits growth when added to such a medium. The thiazole moiety of thiamine, but not the pyrimidine, is also inhibitory, but less so than thiamine itself.Growth inhibition by thiamine is prevented by vitamin B₆. At low concentrations of thiamine, the amount of vitamin B₆ required for growth increases with the thiamine concentration; at concentrations of thiamine above 1 μg./10 ml. the vitamin B₆ requirement for growth remains essentially constant. Since these higher concentrations of thiamine have been used in methods that utilize this organism for determination of vitamin B₆ (1,2), the validity of these methods is confirmed.In the presence of thiamine, growth was also permitted by additions of the thiamine antagonist, neopyrithiamine. In this case, however, the relationship was fully competitive; i.e., the amount of neopyrithiamine required for growth increased regularly with the thiamine concentration. At concentrations considerably higher than those required for growth, neopyrithiamine again inhibited growth, and this inhibition was prevented by an increase in the thiamine concentration. Thus neopyrithiamine acts by lowering the effective thiamine concentration to subinhibitory levels; if excessive amounts are used, it prevents essential metabolic functions of thiamine and itself becomes toxic. The mechanism by which vitamin B₆ prevents thiamine toxicity is not known.The appearance of a requirement for certain growth factors because of inhibitory effects of other metabolically important compounds, rather than because of an intrinsic inability of the organism to synthesize the growth factor, may be much more common than the few recorded instances of this phenomenon indicate.     

Utilization of n-Paraffins and Vitamin B6 Production by Pichia guilliermondii Wickerham

The accumulation of vitamin B₆ by Pichia guilliermondii Wickerham NK–2 strain grown on hydrocarbon was investigated. Ammonium acetate was more effective than other nitrogen sources tested. Satisfactory utilization by the yeast strain was observed in n-alkanes of C₁₀–C₁₈, and n-pentadecane was the best for vitamin B₆ production. Vitamin B₆ was excreted in the cultural broth mainly in the form of pyridoxal, The maximal vitamin B₆ production was approximately 25 mg per liter of the culture broth.     

Selective induction of a giant puff in Drosophila hydei by vitamin B6 and derivatives

Vitamin B₆ induces in vivo as well as in vitro the appearance of a puff at region 2–48C in Drosophila hydei. At concentrations of 10^–2 M or lower, region 2–48C is the only region responding to vitamin B₆ provided that oligomycin is present in the incubation medium. Pyridoxal phosphate and pyridoxamine phosphate supplied to medium containing oligomycin induce upon incubation of salivary glands a larger puff at 2–48C. — Puff 2–48C produces large quantities of a unique RNP-product; globular 140–220 Å particles which aggregate to stable complexes of 0.1–0.2 in diameter. Upon continuous in vitro incubation with vitamin B₆, puff 2–48C becomes loaded with these aggregates which have never been observed in any other puff of Drosophila hydei.     

Regulation of ornithine decarboxylase synthesis. Effect of a nutritional deficiency of vitamin B6

In vitamin B₆ deficiency there is an increase in the activity of the pyridoxal phosphate dependent enzyme ornithine decarboxylase. In the rat liver: the apoenzyme and holoenzyme activity increased 1.6 and 4 fold respectively. Concomitantly, putrescine and spermidine concentrations were halved. The lack of correspondence between product concentration and enzymic activity suggests a control mechanism other than ornithine decarboxylase activity.     

Polyamine biosynthesis and vitamin B6 deficiency Evidence for pyridoxal phosphate as coenzyme for S-adenosylmethionine decarboxylase

Extracts of liver from vitamin B₆-deficient rats had only 50% of the S-adenosylmethionine decarboxylase activity of extracts of liver from control rats when assayed with no exogenous pyridoxal phosphate. When pyridoxal phosphate was included in the reaction mixture, both extracts exhibited the same activity, indicating that pyridoxal phosphate is the coenzyme for S-adenosylmethionine decarboxylase. There was no similar decreased activity in extracts of brain from vitamin B₆-deficient rats.The activity of the pyridoxal phosphate-dependent enzyme, ornithine decarboxylase, was increased in extracts of liver from vitamin B₆-deficient rats: 1.6-fold when assayed with no pyridoxal phosphate and 4-fold when assayed with pyridoxal phosphate.The concentrations of putrescine and spermidine were decreased 50% in liver of vitamin B₆-deficient animals, but only putrescine was decreased in brain. Putreanine was barely detectable in liver of vitamin B₆-deficient animals, but was unchanged in brain.     

Studies on the vitamin B12 auxotrophy of Rhodocyclus purpureus and two other vitamin B12-requiring purple nonsulfur bacteria

The vitamin B₁₂ requirement of Rhodocyclus purpureus 6770, Rhodospirillum tenue 1/67, and Rhodopseudomonas palustris G 53/2 was determined. A wide variety of biogenetic precursors of the vitamin including cobinamide, cobyric acid, cobinic acid and several partially amidated cobyrinic acids showed growth-promoting activity in all three strains. In R. purpureus vitamin B₁₂ could even be substituted by cobyrinic acid which is the first cobalt-containing precursor of vitamin B₁₂ so far established. Neither methionine, deoxynucleosides, dimethylbenzimidazole nor increased amounts of cobalt could replace vitamin B₁₂ as growth factor.Cupribalamin, which is a strong antimetabolite of vitamin B₁₂ in Escherichia coli 113-3 and Lactobacillus leichmannii ATCC 7830, exhibited only a weak antagonistic effect on growth of R. purpureus and R. tenue. Growth of R. palustris was not inhibited by cupribalamin. The cells of all three strains were shown to contain metal-free corrinoids in addition to cobalt-containing corrinoids. The principal products were identified as 5-deoxyadenosylcobalamin and hydrogenobalamin, the metal free analogue of vitamin B₁₂. The latter does not originate from the vitamin by removal of cobalt but is de novo biosynthesized as could be demonstrated in the case of R. purpureus by a labelling experiment with [¹³C] methyl-l-methionine.     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Inflammation causes tissue-specific depletion of vitamin B<Subscript>6</Subscript>

Previously we observed strong and consistent associations between vitamin B₆ status and several indicators of inflammation in patients with rheumatoid arthritis. Clinical indicators, including the disability score, the length of morning stiffness, and the degree of pain, and biochemical markers, including the erythrocyte sedimentation rate and C-reactive protein levels, were found to be inversely correlated with circulating vitamin B₆ levels. Such strong associations imply that impaired vitamin B₆ status in these patients results from inflammation. In the present study we examined whether inflammation directly alters vitamin B₆ tissue contents and its excretion in vivo. A cross-sectional case-controlled human clinical trial was performed in parallel with experiments in an animal model of inflammation. Plasma and erythrocyte and pyridoxal 5'-phosphate concentrations, urinary 4-pyridoxic acid excretion, and the activity coefficient of erythrocyte aspartate aminotransferase were compared between patients and healthy subjects. Adjuvant arthritis was induced in rats for investigating hepatic and muscle contents as well as the urinary excretion of vitamin B₆ during acute and chronic inflammation. Patients with rheumatoid arthritis had low plasma pyridoxal 5'-phosphate compared with healthy control subjects, but normal erythrocyte pyridoxal 5'-phosphate and urinary 4-pyridoxic acid excretion. Adjuvant arthritis in rats did not affect 4-pyridoxic acid excretion or muscle storage of pyridoxal 5'-phosphate, but it resulted in significantly lower pyridoxal 5'-phosphate levels in circulation and in liver during inflammation. Inflammation induced a tissue-specific depletion of vitamin B₆. The low plasma pyridoxal 5'-phosphate levels seen in inflammation are unlikely to be due to insufficient intake or excessive vitamin B₆ excretion. Possible causes of decreased levels of vitamin B₆ are discussed.